Neuregulin induces GABA(A) receptor subunit expression and neurite outgrowth in cerebellar granule cells

Abstract

Neuregulin (NRG), a growth and differentiation factor that signals via erbB receptor tyrosine kinases, has been shown to have biological effects in both the CNS and the peripheral nervous system. We report here that erbB4 is expressed in mature cerebellar granule cells, where it appears to be concentrated at the granule cell postsynaptic terminals. We also show that one form of NRG, Ig-NRG, plays a crucial role in aspects of cerebellar granule cell development in vitro. First, Ig-NRG treatment of granule cells in culture selectively induces the expression of the GABA(A) receptor beta2 subunit. This increase in subunit expression is paralleled by an increase in functional GABA(A) receptors. In contrast to its effects on GABA(A) receptor subunit expression, Ig-NRG does not upregulate NMDA receptor N2B and N2C subunit expression. Second, we demonstrate that Ig-NRG also enhances neurite outgrowth from cultured granule cells. Ig-NRG does not, however, act as a survival factor for the granule cells. We have compared the effect of Ig-NRG with the effects of brain-derived neurotrophic factor (BDNF), a neurotrophin that exerts specific effects on granule cells in culture, and found that BDNF does not mimic the effects of Ig-NRG on GABA(A) receptor subunit expression. Our results show that Ig-NRG has specific effects on granule cell development and maturation and may regulate these processes in vivo.

Fig. 1.

erbB4 and synaptophysin immunoreactivity colocalize at cerebellar glomeruli. A,Top, Parasagittal sections of adult cerebellum were stained with a monoclonal antibody to the synaptic vesicle protein synaptophysin (Oregon green secondary antibody; left) and polyclonal antibodies to erbB4 (0618) and visualized with a Cy3 secondary antibody (right). Bottom, The two top images are superimposed (right), and a bright-field image of the section is shown (left).B,Left, Middle, Confocal images were obtained at higher magnification of the granule cell layer from adult cerebellum stained as described above (synaptophysin,green; erbB4, red). Right, Colocalization of the two proteins is shown by the superimposition of the two images. GL, Granule cell layer; WM, white matter. Scale bars: A, 20 μm; B, 50 μm.

Fig. 2.

Granule cells in culture express erbB4 and respond to Ig-NRG. A, Granule cells express the erbB4 receptor.Top, Granule cells at 2 DIV were stained with a polyclonal anti-erbB4 antibody and a Cy3 secondary antibody. Immunoreactivity was present in the cell bodies and processes of the cells. Bottom, No staining was observed when the erbB4 antibody was preincubated with a cognate peptide.B, Ig-NRG induces p180 tyrosine phosphorylation in granule cells. Granule cells at 2 DIV were stimulated with 1 nm Ig-NRG or vehicle (control) for 5 min. Lysates were resolved by 5% SDS-PAGE, and the blot was probed with an anti-phosphotyrosine antibody. The Ig-NRG-induced tyrosine-phosphorylated band is indicated by the arrow.C, Ig-NRG induces CREB phosphorylation in cultured granule cells. Serum-starved granule cells were treated with Ig-NRG (1 nm for 20 min), fixed, and stained with a phospho-CREB antibody followed by peroxidase reaction. There were significantly more labeled cells in the Ig-NRG-treated cultures (cells appeardark). Scale bars: A, 40 μm;C, 50 μm.

Fig. 3.

Ig-NRG is not a survival factor for granule cells. A, Granule cells in minimal medium were treated with 1 nm Ig-NRG, 5 nm Ig-NRG, 20 ng/ml BDNF, or vehicle (control) for 2 d. Cell survival was then measured by the MTT assay, and the reaction product was analyzed spectrophotometrically. The graph shows an average OD for four independent experiments. Cell survival was significantly increased only by BDNF treatment (BDNF, *p < 0.0001; 1 nm NRG, p = 0.10; 5 nm NRG,p = 0.15). B, Granule cells were treated with 1 nm Ig-NRG, 5 nm Ig-NRG, 20 ng/ml BDNF, or vehicle (control) for 24 hr. Cells were then fixed and stained with Hoechst dye, and the percentage of apoptotic cells was quantified. BDNF but not Ig-NRG treatment significantly reduced the percent of apoptotic cells compared with control (BDNF, *p = 0.014; 1 nm Ig-NRG, p = 0.71; 5 nm Ig-NRG, p = 0.93).

Fig. 4.

Ig-NRG enhances neurite outgrowth from granule cell reaggregates. A, Granule cell reaggregates were treated with vehicle (control), 1 nm Ig-NRG, or 5 nm Ig-NRG for 22 hr. Cultures were then fixed and stained with an antibody to class III β-tubulin and visualized with a Cy3 secondary antibody. Scale bar, 100 μm. B, Ig-NRG and BDNF significantly increase neurite outgrowth from granule cell reaggregates. Reaggregates were treated with 1 nm Ig-NRG, 5 nm Ig-NRG, or 20 ng/ml BDNF and stained with a class III β-tubulin antibody, and neurite length was analyzed. The graph shows a percentage increase in neurite length above control (p < 0.0001 for all three treatments).

Fig. 5.

Ig-NRG treatment upregulates GABA_Areceptor β2 subunit mRNA expression. A,GABA_A subunit mRNA levels were examined in control and Ig-NRG-treated granule cells (2 DIV). The graph shows an average percentage of change in mRNA level relative to control for five independent experiments. Although levels of GABA_A β2 mRNA increased significantly in response to Ig-NRG (**p= 0.02), there was no significant effect on the levels of GABA_A α1 (p = 0.14), GABA_A γ2 (p = 0.96), or NMDA NR2B (p = 0.50) subunit mRNAs.B, BDNF (50 ng/ml) does not induce an upregulation of mRNA for β2 (p = 0.35), α1 (p = 0.50), or γ2 (p = 0.14) GABA_A subunits. The data represent an average of three independent experiments.

Fig. 6.

Ig-NRG increases functional GABA_Areceptors in cultured granule cells. A, Representative whole-cell current responses to varying concentrations of GABA (10, 25, 50, 75, 100, and 150 μm) in a granule cell that was treated with Ig-NRG (filledcircles) and in a control cell (opencircles). Ig-NRG (3 nm) was added to the culture medium at the time of plating, and the GABA responses were assayed on 3 and 4 DIV. Inset, Superimposed computer-generated current traces recorded from the same cells (top, Ig-NRG-treated cell; bottom,control cell) used to generate the dose–response curves.B, Histogram representing the binned distribution of the amplitude of maximal current response to GABA (125 or 150 μm) recorded in granule cells from Ig-NRG-treated (n = 20; solidbars) and control (n = 22; openbars) granule cells. A greater percentage of cells examined in the Ig-NRG-treated cultures are found in the bins representing higher maximal current response amplitudes, and the mean response amplitude is significantly higher than that in control cells (Ig-NRG, 0.34 ± 0.05 nA; control, 0.16 ± 0.03 nA;p < 0.005).